1. Field of the Invention
This invention concerns a device for measuring an amount of gas evolved by cultured cells, and more particularly concerns a radiorespirometer that measures .sup.14 CO.sub.2 production for studying the metabolism of cultured cells.
2. General Discussion of the Background
Oxidation rates of various metabolic pathways of intact cells are determined by measuring .sup.14 CO.sub.2 released from cultured cells. The activity of pyruvate dehydrogenase, for example, can be determined by culturing cells in the presence of radiolabeled [--.sup.14 C]-pyruvate, denaturing cells by acidification with HClO.sub.4, and collecting liberated .sup.14 CO.sub.2 from decarboxylated pyruvate in a vial containing a material that sequesters the .sup.14 CO.sub.2. The amount of liberated .sup.14 CO.sub.2 is then quantitated by placing the vial in a liquid scintillation counter to determine the amount of radioactivity that is present, which in turn indicates the amount of .sup.14 CO.sub.2 that was evolved.
Previous radiorespirometers, and other measurement methods, have involved complex and awkward devices that limit the amount of data that can be collected. Some prior methods require that cells be removed from a petri dish by scraping them from the culture surface, which mechanically injures the cells and disturbs their normal physiology. In other methods, a non-cellular extract is obtained from the cells, and the extract is used to measure enzymatic production of .sup.14 CO.sub.2 from .sup.14 C substrate. Producing non-cellular extracts requires cell disruption that interferes with normal cell metabolism. The resulting data may be biased by the metabolic contribution of damaged cell material.
An example of a previous radiorespirometer was described by Ross et al. in Analytical Biochemistry 112:378-386 (1981). This radiorespirometer is an airtight chamber created by placing a culture plate in a well and clamping a stainless steel cover over the well. A carrier gas is bubbled through the growth medium in the dish to entrain .sup.14 CO.sub.2 and remove it from the culture chamber. The radioactive gas is then taken to an external collection chamber where a CO.sub.2 trapping agent (Oxisorb II) collects the gas in a scintillation vial. The vial is subsequently placed in a liquid scintillator to measure the amount of radioactivity present in the sample. The usefulness of this design is limited by its complexity and the requirement for many airtight chambers.
Another approach to quantitative measurement of substrate oxidation was disclosed in Spahr et al., J. Mol. Cell Cardiol. 21:175-185 (1989), where degradation and oxidation of substrates were estimated using U-.sup.14 C-labeled glucose, lactate and palmitate. Each petri dish containing attached cells was placed in individual airtight chambers and the substrate oxidation was terminated by injecting HCl through a rubber stopper in the cover of an airtight chamber. Radioactive carbon dioxide was then trapped in a KOH solution contained in a reservoir within each incubation chamber.
Shaw and Boder described another method in J. Mol. Cell. Cardiol. 4:485-493 (1972) in which evolution of .sup.14 CO.sub.2 from growing myocytes was measured in a stoppered flask by suspending a plastic collector cup from the rubber stopper of the flask. Although measurements on intact cultured cells can be obtained with such a device, culturing cells in flasks requires a larger number of cells than are required in smaller containers. The number of flasks containing the cells prepared from the same isolation is therefore small, which limits the number of experimental points generated for the cell sample. In addition, many non-malignant cells will not grow or survive in a culture flask.
It is accordingly an object of the present invention to provide an improved simple device in which evolution of gas from living cells can be detected without requiring a complicated apparatus.
It is yet another object of the invention to provide such an improved device that allows many cell cultures from a given isolation to be simultaneously studied, thereby generating larger volumes of data.
Yet another object of the invention is to provide such an improved device in which studies can be performed on physiologically and biochemically intact attached cells.
Finally, it is an object of the invention to provide such an improved device in which the accuracy of data is improved by reducing the metabolic contribution of damaged cell materials.
These and other objects of the invention will be understood more clearly by reference to the following detailed description and drawings.